Non-naturally occuring porcine reproductive and respiratory syndrome virus (prrsv) and methods of using

ABSTRACT

A non-naturally occurring porcine reproductive and respiratory syndrome virus (PRRSV) is provided herein, and methods of making and using the non-naturally occurring PRRSV also are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/054,327 filed Aug. 3, 2018, which is a continuation application ofU.S. patent application Ser. No. 15/127,931 filed on Sep. 21, 2016,which is a U.S. National Application from PCT Application No.PCT/IB2015/052214 filed on Mar. 25, 2015, which claims the benefit ofpriority under 35 U.S.C. § 119(e) to U.S. Application No. 61/968,465,filed Mar. 21, 2014.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under 2013-31100-06031,2012-31100-06031, and 2008-55620-19132 awarded by United StatesDepartment of Agriculture, National Institute of Food and Agriculture.The government has certain rights in the invention.

TECHNICAL FIELD

This disclosure generally relates to a non-naturally occurring porcinereproductive and respiratory syndrome virus (PRRSV) and methods ofusing.

BACKGROUND

Current porcine reproductive and respiratory syndrome virus (PRRSV)vaccines are not adequately effective for control and eradication ofporcine reproductive and respiratory syndrome (PRRS). The mainlimitation of the current PRRSV vaccines is their sub-optimal coverageagainst divergent PRRSV strains. Thus far, all commercial PRRSV vaccinesare formulated using natural PRRSV strains, but the substantial geneticvariation among the PRRSV strains is the biggest obstacle for thedevelopment of a broadly protective PRRSV vaccine.

SUMMARY

This disclosure provides a non-naturally occurring porcine reproductiveand respiratory syndrome virus (PRRSV) and methods of making and usingthe non-naturally occurring PRRSV.

A PRRSV-CON nucleic acid is provided, where the nucleic acid has atleast 50% sequence identity (e.g., at least 75%, at least 95%, or atleast 99% sequence identity) to SEQ ID NO:1. In some embodiment, thenucleic acid has the sequence shown in SEQ ID NO:1. A virus particlecomprising the PPRSV-CON nucleic acid described herein. A compositioncomprising the PPRSV-CON nucleic acid described herein and apharmaceutically acceptable carrier. A composition comprising the virusparticle described herein and a pharmaceutically acceptable carrier. Thecompositions described herein, further comprising an adjuvant.

A PRRSV-CON nucleic acid also is provided, where the nucleic acid has atleast 95% (e.g., at least 99%) sequence identity to a sequence selectedfrom the group consisting of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42. In some embodiments,the nucleic acid has a sequence selected from the group consisting ofSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, and 42. In some embodiments, the nucleic acid encodes,respectively, a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43. A virus particlecomprising the PPRSV-CON nucleic acid described herein. A compositioncomprising the nucleic acid described herein and a pharmaceuticallyacceptable carrier. A composition comprising the virus particledescribed herein and a pharmaceutically acceptable carrier. Thecomposition described herein, further comprising an adjuvant.

A PRRSV-CON polypeptide is provided, where the polypeptide has at least95% (e.g., at least 99%) sequence identity to a sequence selected fromthe group consisting of SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43. In some embodiments, thepolypeptide has a sequence selected from the group consisting of SEQ IDNO:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41 and 43. In some embodiments, the polypeptide is encoded by anucleic acid, respectively, having a sequence selected from the groupconsisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, or 42. A virus particle comprising thePPRSV-CON polypeptide described herein. A composition comprising thepolypeptide described herein and a pharmaceutically acceptable carrier.A composition comprising the virus particle described herein and apharmaceutically acceptable carrier. The composition described herein,further comprising an adjuvant.

A method for eliciting an immune response to PPRSV in a porcine isprovided. Such a method typically includes administering, to a porcine:(i) an effective amount of any of the nucleic acids described herein;(ii) an effective amount of any of the polypeptides described herein;(iii) an effective amount of any of the virus particles describedherein; or (iv) an effective amount of any of the compositions describedherein. Representative routes of administration include, withoutlimitation, intramuscularly, intraperitoneally, and orally.

A method for treating or preventing PPRS in a porcine is provided. Sucha method typically includes administering, to a porcine: (i) aneffective amount of any of the nucleic acids described herein; (ii) aneffective amount of any of the polypeptides described herein; (iii) aneffective amount of any of the virus particles described herein; or (iv)an effective amount of any of the compositions described herein.Representative routes of administration include, without limitation,intramuscularly, intraperitoneally, and orally.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods and compositions of matter belong. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the methods and compositionsof matter, suitable methods and materials are described below. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1B, FIG. 1A is a phylogenetic tree constructed from a set of 60PRRSV full-genome sequences. These 60 PRRSV genomes are classified into4 sub-groups. The locations of the viruses involved in thecross-protection experiments are indicated by the arrows. FIG. 1B is agraph showing the genetic distances among natural PRRSV strains and thegenetic distance from the PRRSV-CON described herein to the naturalPRRSV strains. The lower and upper boundaries of the box indicate the25th and 75th percentile respectively. The solid line within the boxrepresents the median. Whiskers above and below the box indicate theminimum and maximum of the data.

FIGS. 2A-2D show the generation and characterization of the PRRSV-CONvirus. FIG. 2A is a schematic showing the strategy to construct thePRRSV-CON full-genome cDNA clone. The upper half of FIG. 2A depicts theschematic representation of the viral genome, together with the uniquerestriction enzyme sites used for cloning purposes. The horizontal blacklines, with the letters A-D on top, represent the DNA fragments thatwere synthesized. The numbers inside the parenthesis below the linesindicate the length (in nucleotides) of each corresponding fragments.ΦT7 represents the T7 RNA polymerase promoter. Individual DNA fragmentsof the genome were sequentially inserted into the shuttle vector (shownin the lower half of Panel (A)) in the order of fragment A to fragmentD. FIG. 2B are photographs showing the reactivity of the indicatedviruses with different PRRSV-specific monoclonal antibodies. MARC-145cells were mock infected or infected with PRRSV-CON or PRRSV wild typestrain, FL12. At 48 hours post-infection, the cells were stained withantibodies specific to the viral nucleocapsid protein (N protein; bottomrow of photographs) or to the viral nonstructural protein 1 beta (nsp1b;top row of photographs). FIG. 2C shows the plaque morphology of theviruses in MARC-145 cells. FIG. 2D shows a multiple step growth curve.MARC-145 cells were infected with the indicated viruses at amultiplicity of infection (MOI) of 0.01. At different timepointspost-infection (p.i.), culture supernatant was collected and viral titerwas determined by titration on MARC-145 cells.

FIGS. 3A-3D contain data demonstrating replication of the PRRSV-CON inpigs. FIG. 3A shows the rectal temperature measured daily from 1 daybefore infection to 13 days post-infection (days p.i.). FIG. 3B showsthe average daily weight gain (ADWG) within 14 days after inoculation.FIG. 3C shows the viremia levels, determined by a commercial, universalRT-qPCR (Tatracore Inc., Rockville, Md.). FIG. 3D shows the levels ofantibody response after inoculation, determined by IDEXX ELISA; thehorizontal dotted line indicates the cut-off of the assay.

FIGS. 4A-4D contains data demonstrating cross-protection provided by thePRRSV-CON described herein against the PRRSV-strain, MN-184. FIG. 4Ashows the average daily weight gain (ADWG) within 15 days afterchallenge-infection. FIG. 4B shows the viremia levels after challengedetermined by a commercial, universal RT-qPCR (Tetracore Inc.,Rockville, Md.). FIG. 4C shows total viral RNA levels in differenttissues collected at 15 days post-challenge as determined by acommercial, universal RT-qPCR (Tetracore Inc., Rockville, Md.). FIG. 4Dshows the MN-184-specific RNA levels as determined by a differentialRT-qPCR developed in-house.

FIGS. 5A-5D contain data demonstrating cross-protection against PRRSVstrain, 16244B. FIG. 5A shows the average daily weight gain (ADWG)within 15 days after challenge-infection. FIG. 5B shows the viremialevels after challenge infection determined by a commercial, universalRT-qPCR (Tetracore Inc., Rockville, Md.). FIG. 5C shows total viral RNAlevels in different tissues collected at 15 days post-challenge asdetermined by a commercial, universal RT-qPCR (Tetracore Inc.,Rockville, Md.). FIG. 5D shows the 16244B-specific RNA levels asdetermined by a differential RT-qPCR developed in-house.

DETAILED DESCRIPTION

A non-naturally occurring porcine reproductive and respiratory syndromevirus (PRRSV) genome was designed using a large set of genomic sequencesof PRRSV isolates, which represents the widest genetic diversity ofPRRSV strains circulating in U.S. swine herds. The non-naturallyoccurring PRRSV genome was designed so that it has a high degree ofgenetic similarity to the PRRSV field-isolates studied when compared toany single, naturally occurring PRRSV strain.

Porcine reproductive and respiratory syndrome (PRRS) is one of the mosteconomically important diseases in swine. Clinical signs of the diseaseinclude reproductive failure in pregnant sows and respiratory disorderin young pigs. The disease is more severe when animals are co-infectedwith other pathogens. The annual loss to the US swine industry wasestimated to be about $560 million in 2005 and about $640 million in2011.

The causative agent of PRRS is an RNA virus named PRRS virus (PRRSV).PRRSV is classified into two major genotypes: European (Type 1) andNorth American (Type 2). There is limited cross-protection between thesetwo genotypes. Considerable genetic variation exists among PRRSVisolates within each of these genotypes. Importantly, genetic divergencehas been shown to occur when a PRRSV strain is serially passed from pigto pig. This leads to co-circulation of multiple PRRSV variants withinone herd or even within one animal that is persistently infected withPRRSV.

PRRSV vaccines have been in use since 1994. There are two types of PRRSVvaccines currently available in the market; modified-live andinactivated vaccines. In addition, several subunit vaccines againstPRRSV are being tested in different laboratories worldwide, but nonehave been licensed for clinical application. Currently, PRRSV vaccinesare prepared using naturally occurring PRRSV strains as the vaccineimmunogens. The current PRRSV vaccines are not adequately effective forcontrol and eradication of PRRS; they provide acceptable levels ofhomologous protection but they fail to provide consistent heterologouscross-protection. Extensive genetic diversity among PRRSV isolates isthe main reason behind the sub-optimal heterologous protection of thecurrent PRRSV vaccines.

The non-naturally occurring PRRSV-CON described herein confers superiorcross-protective against different heterologous PRRSV strains, ascompared to the PRRSV wild type strain FL12. Thus, the PRRSV-CONdescribed herein can be used to formulate a universal PRRSV vaccine. Inaddition, the PRRSV-CON described herein provides an important tool tostudy the mechanism of heterologous protection against divergent PRRSVstrains.

Nucleic Acids and Polypeptides

The PRRSV genome encodes at least 22 proteins; 14 non-structuralproteins and 8 structural proteins. A nucleic acid is provided hereinthat encodes for a non-naturally occurring PRRSV. See SEQ ID NO:1 forthe genomic sequence of PRRSV-CON. The non-naturally occurring PRRSVdescribed herein possesses the highest degree of genetic identity withthe naturally occurring PRRSV isolates. The PRRSV-CON genomic nucleicacid provided herein (i.e., SEQ ID NO:1) encodes for a number ofdifferent polypeptides. For example, the nucleic acid sequence shown inSEQ ID NO:2 encodes for the polypeptide sequence having the amino acidsequence shown in SEQ ID NO:3; the nucleic acid sequence shown in SEQ IDNO:4 encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:5; the nucleic acid sequence shown in SEQ ID NO:6encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:7; the nucleic acid sequence shown in SEQ ID NO:8encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:9; the nucleic acid sequence shown in SEQ ID NO:10encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:11; the nucleic acid sequence shown in SEQ ID NO:12encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:13; the nucleic acid sequence shown in SEQ ID NO:14encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:15; the nucleic acid sequence shown in SEQ ID NO:16encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:17; the nucleic acid sequence shown in SEQ ID NO:18encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:19; the nucleic acid sequence shown in SEQ ID NO:20encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:21; the nucleic acid sequence shown in SEQ ID NO:22encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:23; the nucleic acid sequence shown in SEQ ID NO:24encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:25; the nucleic acid sequence shown in SEQ ID NO:26encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:27; the nucleic acid sequence shown in SEQ ID NO:28encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:29; the nucleic acid sequence shown in SEQ ID NO:30encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:31; the nucleic acid sequence shown in SEQ ID NO:32encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:33; the nucleic acid sequence shown in SEQ ID NO:34encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:35; the nucleic acid sequence shown in SEQ ID NO:36encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:37; the nucleic acid sequence shown in SEQ ID NO:38encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:39; the nucleic acid sequence shown in SEQ ID NO:40encodes for the polypeptide sequence having the amino acid sequenceshown in SEQ ID NO:41; and the nucleic acid sequence shown in SEQ IDNO:42 encodes for the polypeptide sequence having the amino acidsequence shown in SEQ ID NO:43.

As used herein, nucleic acids can include DNA and RNA, and includesnucleic acids that contain one or more nucleotide analogs or backbonemodifications. A nucleic acid can be single stranded or double stranded,which usually depends upon its intended use. Nucleic acids andpolypeptides that differ from SEQ ID NOs:1-43 also are provided. Nucleicacids that differ in sequence from SEQ ID NO:1 or any of SEQ ID NOs:2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,or 42 can have at least 80% sequence identity (e.g., at least 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:1 or any of SEQ IDNOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, or 42. Polypeptides that differ in sequence from any of SEQ IDNOs:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41 or 43, can have at least 80% sequence identity (e.g., at least81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity) to any of SEQ ID NOs:3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or43.

In calculating percent sequence identity, two sequences are aligned andthe number of identical matches of nucleotides or amino acid residuesbetween the two sequences is determined. The number of identical matchesis divided by the length of the aligned region (i.e., the number ofaligned nucleotides or amino acid residues) and multiplied by 100 toarrive at a percent sequence identity value. It will be appreciated thatthe length of the aligned region can be a portion of one or bothsequences up to the full-length size of the shortest sequence. It alsowill be appreciated that a single sequence can align with more than oneother sequence and hence, can have different percent sequence identityvalues over each aligned region.

The alignment of two or more sequences to determine percent sequenceidentity can be performed using the computer program ClustalW anddefault parameters, which allows alignments of nucleic acid orpolypeptide sequences to be carried out across their entire length(global alignment). Chenna et al., 2003, Nucleic Acids Res.,31(13):3497-500. ClustalW calculates the best match between a query andone or more subject sequences, and aligns them so that identities,similarities and differences can be determined. Gaps of one or moreresidues can be inserted into a query sequence, a subject sequence, orboth, to maximize sequence alignments. For fast pairwise alignment ofnucleic acid sequences, the default parameters can be used (i.e., wordsize: 2; window size: 4; scoring method: percentage; number of topdiagonals: 4; and gap penalty: 5); for an alignment of multiple nucleicacid sequences, the following parameters can be used: gap openingpenalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes.For fast pairwise alignment of polypeptide sequences, the followingparameters can be used: word size: 1; window size: 5; scoring method:percentage; number of top diagonals: 5; and gap penalty: 3. For multiplealignment of polypeptide sequences, the following parameters can beused: weight matrix: blosum; gap opening penalty: 10.0; gap extensionpenalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro,Ser, Asn, Asp, Gln, Glu, Arg, and Lys; and residue-specific gappenalties: on. ClustalW can be run, for example, at the Baylor Collegeof Medicine Search Launcher website or at the European BioinformaticsInstitute website on the World Wide Web.

Changes can be introduced into a nucleic acid molecule (e.g., SEQ IDNO:1 or any of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, or 42), thereby leading to changes inthe amino acid sequence of the encoded polypeptide (e.g., SEQ ID NOs:3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41or 43). For example, changes can be introduced into nucleic acid codingsequences using mutagenesis (e.g., site-directed mutagenesis,PCR-mediated mutagenesis) or by chemically synthesizing a nucleic acidmolecule having such changes. Such nucleic acid changes can lead toconservative and/or non-conservative amino acid substitutions at one ormore amino acid residues. A “conservative amino acid substitution” isone in which one amino acid residue is replaced with a different aminoacid residue having a similar side chain (see, for example, Dayhoff etal. (1978, in Atlas of Protein Sequence and Structure, 5(Suppl.3):345-352), which provides frequency tables for amino acidsubstitutions), and a non-conservative substitution is one in which anamino acid residue is replaced with an amino acid residue that does nothave a similar side chain.

As used herein, an “isolated” nucleic acid molecule is a nucleic acidmolecule that is free of sequences that naturally flank one or both endsof the nucleic acid in the genome of the organism from which theisolated nucleic acid molecule is derived (e.g., a cDNA or genomic DNAfragment produced by PCR or restriction endonuclease digestion). Such anisolated nucleic acid molecule is generally introduced into a vector(e.g., a cloning vector, or an expression vector) for convenience ofmanipulation or to generate a fusion nucleic acid molecule, discussed inmore detail below. In addition, an isolated nucleic acid molecule caninclude an engineered nucleic acid molecule such as a recombinant or asynthetic nucleic acid molecule.

As used herein, a “purified” polypeptide is a polypeptide that has beenseparated or purified from cellular components that naturally accompanyit. Typically, the polypeptide is considered “purified” when it is atleast 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dryweight, free from the polypeptides and naturally occurring moleculeswith which it is naturally associated. Since a polypeptide that ischemically synthesized is, by nature, separated from the components thatnaturally accompany it, a synthetic polypeptide is “purified.”

Nucleic acids can be isolated using techniques routine in the art. Forexample, nucleic acids can be isolated using any method including,without limitation, recombinant nucleic acid technology, and/or thepolymerase chain reaction (PCR). General PCR techniques are described,for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler,Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleicacid techniques include, for example, restriction enzyme digestion andligation, which can be used to isolate a nucleic acid. Isolated nucleicacids also can be chemically synthesized, either as a single nucleicacid molecule or as a series of oligonucleotides.

Polypeptides can be purified from natural sources (e.g., a biologicalsample) by known methods such as DEAE ion exchange, gel filtration, andhydroxyapatite chromatography. A polypeptide also can be purified, forexample, by expressing a nucleic acid in an expression vector. Inaddition, a purified polypeptide can be obtained by chemical synthesis.The extent of purity of a polypeptide can be measured using anyappropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

A vector containing a nucleic acid (e.g., a nucleic acid that encodes apolypeptide) also is provided. Vectors, including expression vectors,are commercially available or can be produced by recombinant DNAtechniques routine in the art. A vector containing a nucleic acid canhave expression elements operably linked to such a nucleic acid, andfurther can include sequences such as those encoding a selectable marker(e.g., an antibiotic resistance gene). A vector containing a nucleicacid can encode a chimeric or fusion polypeptide (i.e., a polypeptideoperatively linked to a heterologous polypeptide, which can be at eitherthe N-terminus or C-terminus of the polypeptide). Representativeheterologous polypeptides are those that can be used in purification ofthe encoded polypeptide (e.g., 6xHis tag, glutathione S-transferase(GST))

Expression elements include nucleic acid sequences that direct andregulate expression of nucleic acid coding sequences. One example of anexpression element is a promoter sequence. Expression elements also caninclude introns, enhancer sequences, response elements, or inducibleelements that modulate expression of a nucleic acid. Expression elementscan be of bacterial, yeast, insect, mammalian, or viral origin, andvectors can contain a combination of elements from different origins. Asused herein, operably linked means that a promoter or other expressionelement(s) are positioned in a vector relative to a nucleic acid in sucha way as to direct or regulate expression of the nucleic acid (e.g.,in-frame). Many methods for introducing nucleic acids into host cells,both in vivo and in vitro, are well known to those skilled in the artand include, without limitation, electroporation, calcium phosphateprecipitation, polyethylene glycol (PEG) transformation, heat shock,lipofection, microinjection, and viral-mediated nucleic acid transfer.

Vectors as described herein can be introduced into a host cell. As usedherein, “host cell” refers to the particular cell into which the nucleicacid is introduced and also includes the progeny of such a cell thatcarry the vector. A host cell can be any prokaryotic or eukaryotic cell.For example, nucleic acids can be expressed in bacterial cells such asE. coli, or in insect cells, yeast or mammalian cells (such as Chinesehamster ovary cells (CHO) or COS cells). Other suitable host cells areknown to those skilled in the art.

Nucleic acids can be detected using any number of amplificationtechniques (see, e.g., PCR Primer: A Laboratory Manual, 1995,Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; and U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159;and 4,965,188) with an appropriate pair of oligonucleotides (e.g.,primers). A number of modifications to the original PCR have beendeveloped and can be used to detect a nucleic acid.

Nucleic acids also can be detected using hybridization. Hybridizationbetween nucleic acids is discussed in detail in Sambrook et al. (1989,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sections 7.37-7.57,9.47-9.57, 11.7-11.8, and 11.45-11.57). Sambrook et al. disclosessuitable Southern blot conditions for oligonucleotide probes less thanabout 100 nucleotides (Sections 11.45-11.46). The Tm between a sequencethat is less than 100 nucleotides in length and a second sequence can becalculated using the formula provided in Section 11.46. Sambrook et al.additionally discloses Southern blot conditions for oligonucleotideprobes greater than about 100 nucleotides (see Sections 9.47-9.54). TheTm between a sequence greater than 100 nucleotides in length and asecond sequence can be calculated using the formula provided in Sections9.50-9.51 of Sambrook et al.

The conditions under which membranes containing nucleic acids areprehybridized and hybridized, as well as the conditions under whichmembranes containing nucleic acids are washed to remove excess andnon-specifically bound probe, can play a significant role in thestringency of the hybridization. Such hybridizations and washes can beperformed, where appropriate, under moderate or high stringencyconditions. For example, washing conditions can be made more stringentby decreasing the salt concentration in the wash solutions and/or byincreasing the temperature at which the washes are performed. Simply byway of example, high stringency conditions typically include a wash ofthe membranes in 0.2XSSC at 65° C.

In addition, interpreting the amount of hybridization can be affected,for example, by the specific activity of the labeled oligonucleotideprobe, by the number of probe-binding sites on the template nucleic acidto which the probe has hybridized, and by the amount of exposure of anautoradiograph or other detection medium. It will be readily appreciatedby those of ordinary skill in the art that although any number ofhybridization and washing conditions can be used to examinehybridization of a probe nucleic acid molecule to immobilized targetnucleic acids, it is more important to examine hybridization of a probeto target nucleic acids under identical hybridization, washing, andexposure conditions. Preferably, the target nucleic acids are on thesame membrane.

A nucleic acid molecule is deemed to hybridize to a nucleic acid but notto another nucleic acid if hybridization to a nucleic acid is at least5-fold (e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,50-fold, or 100-fold) greater than hybridization to another nucleicacid. The amount of hybridization can be quantitated directly on amembrane or from an autoradiograph using, for example, a PhosphorImageror a Densitometer (Molecular Dynamics, Sunnyvale, Calif.).

Polypeptides can be detected using antibodies. Techniques for detectingpolypeptides using antibodies include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations and immunofluorescence. Anantibody can be polyclonal or monoclonal. An antibody having specificbinding affinity for a polypeptide can be generated using methods wellknown in the art. The antibody can be attached to a solid support suchas a microtiter plate using methods known in the art. In the presence ofa polypeptide, an antibody-polypeptide complex is formed.

Detection (e.g., of an amplification product, a hybridization complex,or a polypeptide) is usually accomplished using detectable labels. Theterm “label” is intended to encompass the use of direct labels as wellas indirect labels. Detectable labels include enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials.

Methods of Making and Using a PRRSV-CON Virus Particle

Methods of constructing a virus particle from a PRRSV-CON nucleic acidare known in the art and are described herein. As demonstrated herein,the PRRSV-CON described herein self-assembles into particles whenappropriately expressed. The PRRSV-CON can be expressed in vitro or invivo, for example, in a host cell. In some embodiments, a host cell canbe transfected with the PRRSV-CON nucleic acid, or a host cell can beinfected with a PRRSV-CON virus particle. Host cells can be, withoutlimitation, porcine cells (e.g., porcine alveolar macrophage) or Africangreen monkey kidney-derived cells (e.g., MARC-145). Virus particles canbe isolated, for example, by ultracentrifugation.

The PRRSV-CON nucleic acids, polypeptides or virus particles describedherein can be used to generate, enhance or modulate the immune responseof a porcine. Such methods typically include administering a PRRSV-CONnucleic acid, polypeptide or virus particle described herein to aporcine in an amount sufficient to generate an immune response. As usedherein, an “immune response” refers to the reaction elicited in anindividual following administration of a PRRSV-CON nucleic acid,polypeptide or virus particle as described herein. Immune responses caninclude, for example, an antibody response or a cellular response (e.g.,a cytotoxic T-cell response). A PRRSV-CON nucleic acid, polypeptide orvirus particle can be used to prevent PRRS in porcine, e.g., as aprophylactic vaccine, or to establish or enhance immunity to PRRS in ahealthy individual prior to exposure or contraction of PRRS, thuspreventing the disease or reducing the severity of disease symptoms.

Methods for administering a PRRSV-CON nucleic acid, polypeptide or virusparticle to a porcine include, without limitation, intramuscular (i.m.),subcutaneous (s.c.), or intrapulmonary routes. Methods for administeringa PRRSV-CON nucleic acid, polypeptide or virus particle to a porcinealso include, without limitation, intratracheal, transdermal,intraocular, intranasal, inhalation, intracavity, and intravenous (i.v.)administration.

Determining an effective amount of a PRRSV-CON nucleic acid, polypeptideor virus particle depends upon a number of factors including, forexample, whether the antigen is being expressed or administereddirectly, the age and weight of the subject, the precise conditionrequiring treatment and its severity, and the route of administration.Based on the above factors, determining the amount and the dosing (e.g.,the number of doses and the timing of doses) are within the level ofskill of an ordinary artisan.

A composition can include a PRRSV-CON nucleic acid, polypeptide or virusparticle as described herein and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are known in the art and include,for example, buffers (e.g., phosphate buffered saline (PBS), normalsaline, Tris buffer, and sodium phosphate) or diluents. The compositionsdescribed herein can be formulated as an aqueous solution, or as anemulsion, gel, solution, suspension, or powder. See, for example,Remington's Pharmaceutical Sciences, 16th Ed., Osol, ed., MackPublishing Co., Easton, Pa. (1980), and Remington's PharmaceuticalSciences, 19th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.(1995). In addition to a pharmaceutically acceptable carrier, thecompositions described herein also can include binders, stabilizers,preservatives, salts, excipients, delivery vehicles and/or auxiliaryagents.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The invention will be furtherdescribed in the following examples, which do not limit the scope of themethods and compositions of matter described in the claims.

EXAMPLES Example 1—Computational Design of the Artificial PRRSV-CONGenome

Full-genome sequences of 64 PRRSV isolates originating from theMidwestern states (Iowa, Nebraska and Illinois) of the U.S. weresequenced using the Roche 454-GS-FLX sequencing technology. In addition,more than 20 full-genome sequences of PRRSV isolates originating fromthe U.S. were collected from GenBank. After removing redundantsequences, a final set of 60 full-genome sequences of PRRSV wasattained. The 60 PRRSV full-genome sequences were aligned using theMUSCLE program (Edgar RC, 2004, BMC Bioinform., 5:113). After that, aconsensus genome sequence (PRRSV-CON) was generated by selecting themost common nucleotide found at each position of the viral genome, usingthe Jalview program. Phylogenetic analysis shows that the PRRSV-CONgenome locates right at the center of the phylogenetic tree. See FIG.1A. Consequently, the pairwise genetic distance from PRRSV-CON to thenaturally occurring PRRSV strains is significantly shorter than thedistance from any one naturally occurring PRRSV strains to each other(p<0.0001). See FIG. 1B.

Example 2—Generation of an Infectious PRRSV-CON Virus

It is generally difficult to accurately determine the sequence at 5′ and3′ ends of a viral genome. Thus, we realized that the sequences at the5′ and 3′ untranslated regions (UTRs) of the naturally occurring PRRSVgenomes analyzed in Example 1 may not be accurate. To increase thechange of recovering infectious virus, we replaced the 5′ and 3′ UTRs ofthe PRRSV-CON genome with the 5′ and 3′ UTRs of the infectious cDNAclone FL12 (Truong et al., 2004, Virology, 325:308-19). Four DNAfragments, designated A-D, encompassing the entire PRRSV-CON genome,were chemically synthesized by Genscript (Piscataway, N.J.). Each DNAfragment was flanked by a pair of restriction enzyme sites to facilitatethe cloning purposes. The T7 RNA polymerase promoter sequence wasincorporated into fragment D, preceding the viral 5′ end, to facilitatethe in vitro transcription of the viral genome. See FIG. 2A. IndividualDNA fragments were sequentially cloned into the shuttle vector thatcarries the corresponding restriction enzyme site, following the orderfrom fragment A to fragment D. Once the full-length PRRSV-CON cDNA clonewas generated, standard reverse genetics techniques were applied torecover viable PRRSV-CON viruses.

Briefly, the plasmid containing full-length cDNA genome of PRRSV-CON wasdigested with AclI for linearization. The purified, linear DNA fragmentwas used as the template for an in vitro transcription reaction usingthe mMESSAGEmMACHINE Ultra T7 kit (Ambion, Austin, Tex.) to generatefull genome viral RNA transcripts. After that, about 5 μg of thefull-genome RNA transcripts were transfected into MARC-145 cellscultured in a 6-well plate, using the TransIT®-mRNA Transfection Kit(Mirus Bio, Madison, Wis.). Transfected cells were cultured in DMEMcontaining 10% FBS at 37° C., 5% CO2 for up to 6 days. Typically,cytopathic effect (CPE) was observed between day 4 and day 6 aftertransfection. When clear CPE was observed, culture supernatantcontaining the rescued virus was collected and stored in 0.5 mL aliquotsin a 80° C. freezer. See, Truong et al. (2004, supra)

Example 3—In Vitro Characterization of the PRRSV-CON Virus

To study the reactivity with different PRRSV-specific monoclonalantibodies, MARC-145 cells were mock infected or infected with thePRRSV-CON virus or the PRRSV strain FL12. At 48 hours post-infection(p.i.), the cells were immunostained with antibodies specific to theviral nucleocapsid (N) protein or the viral nonstructural protein 1 beta(nsp1b). To study the growth kinetics of the viruses in cell culture,MARC-145 cells were infected with the PRRSV-CON or FL12 at amultiplicity of infection (MOI) of 0.01. At different time-points p.i.,culture supernatant was collected and viral titers were determined bytitration in MARC-145 cells.

The PRRSV-CON virus displays typical in vitro characterizations of anaturally occurring PRRSV strain. It reacts with differentPRRSV-specific monoclonal antibodies including antibodies againstnsp1-betta and N protein (FIG. 2B). It replicates efficiently in cellculture (FIG. 2C), and it is able to form clear and distinct plaquemorphology (FIG. 3D).

Example 4—The PRRSV-CON Virus Can Infect Pigs as Efficiently as theNatural PRRSV Strain

A total of 18 PRRSV-seronegative, 3 week-old pigs were purchased fromthe University of Nebraska research farm. The pigs were randomlyassigned into 3 experimental groups; each group was housed in a separateroom in the Biosecurity Level-2 Animal Research Facilities at UNL,following the regulations established by the Institutional Animal Careand Use Committee. Pigs in group 1 were injected with PBS to act as thecontrol. Pigs in groups 2 and 3 were inoculated intramuscularly with10^(5.0) TCID₅₀ of PRRSV-CON and PRRSV strain FL12, respectively. Thewild-type PRRSV strain, FL12, was included into this study forcomparison purposes. The results are shown in FIG. 3. After infection,both of the PRRSV-CON and FL12-inoculated groups displayed significantlyhigher temperature than PBS-group (FIG. 3A), but there was no differencein temperature between PRRSV-CON-inoculated group and theFL12-inoculated group. Average daily weight gain (ADWG) was measured foreach individual pig during the period of 14 days after infection. Nostatistical difference was observed among the three treatment groups,although pigs in the PRRSV-CON-inoculated group and the FL12-inoculatedgroup tended to have lower ADWG than the PBS group (FIG. 3B). Viremialevels of the PRRSV-CON- and FL12-inoculated groups were almostidentical (FIG. 3C). All pigs in the PRRSV-CON- and FL12-inoculatedgroups were seroconverted by 11 days p.i. The level of antibody responsein the PRRSV-CON-inoculated group was slightly lower than that of theFL12-inoculated group (FIG. 3D). These results demonstrate that thePRRSV-CON can infect the natural host (i.e., pigs) as efficiently as thePRRSV strain, FL12.

Example 5—Evaluation of the Level of Cross-Protection Against PRRSVStrain MN-184 Materials and Methods

A total of 18 PRRSV-seronegative, 3 week-old pigs were purchased fromthe University of Nebraska research farm. The pigs were randomlyassigned into 3 experimental groups; each group was housed in a separateroom in the Biosecurity Level-2 Animal Research Facilities at UNL,following the regulations established by the Institutional Animal Careand Use Committee. Group 1 was injected with PBS and served as thenon-immunization control. Group 2 was immunized by infection,intramuscularly, with PRRSV-CON at the dose of 10^(4.0) TCID₅₀ per pig.Group 3 was immunized by infection, intramuscularly, with the wild-typePRRSV strain, FL12, at the dose of 10^(4.0) TCID₅₀ per pig. See Table 1.At 53 days post-infection (p.i.), all control and immunized pigs werechallenged, intramuscularly, with PRRSV strain MN-184 at a dose of10^(5.0) TCID₅₀. Parameters used to evaluate protection by immunizationwith the PRRSV-CON virus included viremia and viral load in severaldifferent tissues as well as growth performance.

TABLE 1 Experimental Design to Evaluate Level of Cross- ProtectionAgainst PRRSV Strain MN-184 Groups Immunized with Challenged with 1 (n =6) PBS MN-184 2 (n = 6) PRRSV-CON (Sub-group 2) 3 (n = 6_(—) PRRSVstrain FL12

To measure growth performance, each pig was weighed right beforechallenge infection and 15 days post-challenge. Body weight was recordedin pounds. Average daily weight gain (ADWG) was calculated for theperiod of 15 days post-challenge.

To quantitate levels of viremia after challenge infection, blood sampleswere taken before challenge and at days 1, 4, 7, 10, and 15post-challenge. Serum samples were extracted from each individual bloodsamples and stored in a −80° C. freezer. Viremia levels were quantitatedby the Animal Disease Research and Diagnostic Laboratory, South DakotaState University, using the universal RT-qPCR kit (Tetracore Inc.,Rockville, Md.). Results were reported as log10 copy/mL. For statisticalpurposes, samples that had undetected level of viral RNA were assigned avalue of 0 log10 copy/mL.

To quantitate levels of viral load in tissues, pigs were humanelysacrificed and necropsied on day 15 post-challenge. Samples of tonsil,lung, mediastinal lymph node and inguinal lymph node were obtained andkept individually in Whirl-pak® bags. The samples were snap-frozen inliquid nitrogen right after collection. After that, they were stored ina −80° C. freezer. To extract RNA, tissue samples were homogenized inTrizol reagent (Life Technologies, Carlsbad, Calif.) with a ratio of 300mg tissue in 3 mL Trizol reagent. Total RNA was extracted using theRNeasy Mini Kit (Qiagen, Valencia, Calif.) following the manufacturer'sinstruction. RNA concentration was quantitated by the NanoDrop®ND-1000(NanoDrop Technologies, Inc., Wilmington, Del.) and adjusted to a finalconcentration of 200 ng/μL.

It has been well characterized that PRRSV can colonize and persist inlymphoid tissues of infected pigs up to 150 days post-infection. Inthese experiments, the tissue viral load was evaluated at 15 dayspost-challenge, which corresponds to 67 days after the primaryinfection. At that time, it is likely that the pigs in the PRRSV-CON andFL12 groups still contained residual virus of the primary infection.Therefore, we used two different RT-PCR kits to quantify the viral RNAload in tissues: (i) the commercial RT-qPCR kit (Tetracore Inc.,Rockville, Md.) that detects total viral RNA resulting from both theprimary infection and the challenge infection, and (ii) the differentialRT-PCR developed in-house that selectively detects only viral RNA fromchallenge infection. Five μL of each RNA sample (equivalent to 1 μg RNA)was used for each RT-qPCR reaction. Results were reported as log10copy/μg of total RNA. For statistical purposes, samples that hadundetected viral RNA level were assigned a value of 0 log RNA copy/1 μgof total RNA.

Results

The results of growth performance are presented in FIG. 4A. The meanADWG of PBS-, PRRSV-CON- and FL12-immunized groups were 0.3 lbs(SD+/−0.3), 0.9 lbs (SD+/−0.6), and 1.2 lbs (SD+/−0.4), respectively.PRRSV-CON and FL12-immunized groups had greater ADWG than thePBS-immunized group. There was no statistical difference between thePRRSV-CON- and FL12-immunized groups.

The viremia levels after challenge infection are shown in FIG. 4B andTable 2. All pigs in the PBS-immunized group were viremic at alltimepoints tested. The PRRSV-CON-immunized group only had 3 viremicpigs, of which 1 pig was viremic at 2 timepoints (pig #494 at 4 DPC and7 DPC) and 2 pigs were viremic at only one timepoint (pigs #394 and 495at 15 DPC). The remaining 3 pigs in this group (pigs #345, 410 and 459)were not viremic after challenge infection. By contrast, viremia wasdetected in 5 out of 6 pigs in the FL12-immunized group at twotime-points or more after challenge infection. There was only 1 pig inthis group (pig #440) that was not viremic at any time-point tested.Overall, the viremia level of PRRSV-CON-immunized pigs was significantlylower than that in the FL12-immunized group (p<0.05) and thePBS-immunized group (p<0.0001).

The results of total viral RNA quanititated by the universal RT-qPCR kitare shown in FIG. 4C. The PRRSV-CON- and FL12-immunized groups containedsignificantly lower levels of total viral RNA than the PBS-immunizedgroup, regardless of the tissue types tested. However, there was nodifference between the PRRSV-CON- and FL12-immunized groups in term oftotal viral RNA.

The results of MN-184 specific RNA quantitated by the differentialRT-qPCR are shown in FIG. 4D. All pigs in PBS-immunized group carriedMN-184 RNA in their tissues. Four pigs in the FL12-immunized group hadMN-184 RNA in their tonsil and mediastinal lymph node, whereas 5 pigs inthis group had MN-184 RNA in their inguinal lymph node. Remarkably, noneof the pigs in the PRRSV-CON-immunized group had detectable level ofMN-184 RNA in any of the tissue samples tested.

Taken together, these results clearly demonstrate that immunization ofweaning pigs by infection with the non-naturally occurring PRRSV-CONresulted in significantly better cross-protection against challenge withPRRSV strain, MN-184, than did immunization with the PRRSV strain, FL12.

TABLE 2 Viremia After Challenge Infection (log10 copy/mL) Daypost-challenge infection (DPC) Treatment Pig ID 0 DPC 1 DPC 4 DPC 7 DPC10 DPC 15 DPC Group 1 365 0.00 4.94 5.43 5.45 6.79 6.32 (Injected 3890.00 6.26 6.08 5.40 7.60 6.93 (“immunized”) 407 0.00 4.91 6.00 5.86 7.566.75 with PBS) 416 0.00 6.20 6.04 5.20 7.18 6.78 417 0.00 5.18 5.59 4.865.90 6.45 435 0.00 5.83 5.08 5.94 5.57 5.36 Mean 0.00 5.55 5.70 5.456.77 6.43 SD 0.00 0.62 0.40 0.40 0.86 0.57 Group 2 345 0.00 0.00 0.000.00 0.00 0.00 (Immunized by 394 0.00 0.00 0.00 0.00 0.00 2.58 infectionwith 410 0.00 0.00 0.00 0.00 0.00 0.00 PRRSV-CON) 459 0.00 0.00 0.000.00 0.00 0.00 494 0.00 0.00 3.58 5.98 0.00 0.00 495 0.00 0.00 0.00 0.000.00 2.98 Mean 0.00 0.00 0.60 1.00 0.00 0.93 SD 0.00 0.00 1.46 2.44 0.001.44 Group 3 349 0.00 0.00 2.81 2.92 0.00 0.00 (Immunized by 381 0.000.00 0.00 3.04 2.86 0.00 infection with 440 0.00 0.00 0.00 0.00 0.000.00 FL12) 455 0.00 0.00 4.18 4.34 0.00 0.00 487 0.00 3.59 5.28 2.405.60 2.68 507 0.00 2.32 5.56 3.70 0.00 0.00 Mean 0.00 0.99 2.97 2.731.41 0.45 SD 0.00 1.58 2.50 1.50 2.35 1.09

Example 6—Evaluation of the Level of Cross-Protection Against PRRSVStrain 16244B Materials and Methods

The experimental design was the same as described above in Example 5. Atotal of 18 PRRSV-seronegative, 3 week-old pigs purchased from the UNLresearch farm were randomly assigned into 3 experimental groups. Eachgroup was housed in a separate room at the Biosecurity Level-2 AnimalResearch Facilities at UNL, following the regulations established by theInstitutional Animal Care and Use Committee. Group 1 was injected withPBS and acted as the control. Group 2 was immunized, intramuscularly, byinfection with PRRSV-CON at the dose of 10^(4.0) TCID50 per pig. Group 3was immunized, intramuscularly, by infection with the wild type PRRSV,FL12, at the dose of 10^(4.0) TCID50 per pig. See Table 3. One pig ingroup 3 (pig #543) and one pig in group 2 (pig #435) were removed fromthis study on 14 and 23 days after primary infection, respectively, dueto lameness in their legs. At day 52 post-infection (p.i.), all pigswere challenged, intramuscularly, with PRRSV strain 16244B at thechallenge dose of 10^(5.0) TCID50. Parameters used to evaluateprotection by immunization with the PRRSV-CON virus, including viremiaand viral load in various tissues as well as growth performance, weremeasured as described above in Example 5.

TABLE 3 Experimental Design to Evaluate Level of Cross- ProtectionAgainst PRRSV Strain 16244B Groups Immunized with Challenged with 1 (n =6) PBS 16244B 2 (n = 6) PRRSV-CON (sub-group 3) 3 (n = 6) PRRSV strainFL12

Results

The results of growth performance are shown in FIG. 5A. Mean ADWG ofPBS-, PRRSV-CON-, and FL12-immunized groups were 1.1 lbs (SD+/−0.3), 1.6lbs (SD+/−0.1), and 0.8 lbs (SD+/−0.3), respectively. ThePRRSV-CON-immunized group had greater ADWG than the PBS-immunized groupand the FL12-immunized group; whereas the FL12-immunized group was notstatistically different from the PBS-immunized group.

The results of viremia levels after challenge infection are shown inFIG. 5B and Table 4. All pigs in the PBS-immunized group were viremic atall timepoints tested. Two out of 5 pigs in the PRRSV-CON-immunizedgroup (pigs #442 and 445) did not resolve viremia at 52 days afterprimary infection as viral RNA was still detected in their serum samplescollected at this timepoint. After challenge infection, 3 pigs in thePRRSV-CON-immunized group were viremic at only 1 timepoint. Theremaining 2 pigs in this group (pigs #436 and 438) were not viremicthroughout the period of 15 days post-challenge. By contrast, all pigsin the FL12-immunized group resolved viremia by 52 days post-primaryinfection. After challenge infection, all pigs in this group becameviremic. Overall, the viremia level of the PRRSV-CON-immunized group wassignificantly lower than that of the FL12-immunized group (p<0.0001) orthe PBS-immunized group (p<0.0001).

The results of total viral RNA quantitated by the commercial RT-qPCR kit(Tetracore Inc., Rockville, Md.) are shown in FIG. 5C. Both thePRRSV-CON- and FL12-immunized groups contained significantly lowerlevels of total viral RNA than the PBS-immunized group, regardless ofthe tissue types tested. However, there was no statistical differencebetween the PRRSV-CON-immunized group and the FL12-immunized group interms of total viral RNA.

The results of 16244B-specific RNA quantitated by the differentialRT-qPCR are shown in FIG. 5D. All pigs in the PBS- and FL12-immunizedgroups carried 16244B-specific RNA in their tissues, although the levelsof 16244B RNA in the FL12-immunized group was lower than those in thePBS-immunized group. By contrast, only 1 pig in the PRRSV-CON-immunizedgroup carried 16244B-specific RNA in its inguinal lymph node, while theremaining 4 pigs in this group did not carry 16244B-specific RNA.

All together, these results clearly demonstrate that immunization ofweaning pigs by infection with the non-naturally occurring PRRSV-CONresulted in significantly better cross-protection against challenge withPRRSV strain, 16244B, than did immunization with the PRRSV strain, FL12.

TABLE 4 Level of Viremia After Challenge Infection (log10 copy/mL) Daypost-challenge Treatment Pig ID 0 DPC 1 DPC 4 DPC 7 DPC 11 DPC 14 DPCGroup 1 440 0.00 6.62 6.99 6.79 6.15 4.67 (Injected 441 0.00 6.61 6.937.11 5.79 4.81 with PBS) 544 0.00 6.85 6.82 6.96 3.91 5.68 545 0.00 7.117.41 7.11 6.81 5.93 546 0.00 6.74 7.45 7.30 5.67 5.40 547 0.00 6.77 7.517.36 6.73 5.52 Mean 0.00 6.78 7.18 7.11 5.84 5.34 SD 0.00 0.18 0.30 0.211.06 0.50 Group 2 435 Removed from experiment on day 23rd after primary(immunized infection by infection 436 0.00 0.00 0.00 0.00 0.00 0.00 with437 0.00 2.48 0.00 0.00 0.00 0.00 PRRSV-CON) 438 0.00 0.00 0.00 0.000.00 0.00 442 2.81 0.00 0.00 0.00 0.00 2.93 445 3.00 3.32 0.00 0.00 0.000.00 Mean 1.16 1.16 0.00 0.00 0.00 0.59 SD 1.59 1.62 0.00 0.00 0.00 1.31Group 3 439 0.00 4.34 6.78 3.54 2.48 0.00 (immunized 444 0.00 3.04 6.580.00 0.00 0.00 by infection 446 0.00 5.26 4.84 0.00 0.00 0.00 with FL12)526 0.00 2.98 4.40 4.15 0.00 0.00 540 0.00 3.90 4.18 5.08 3.95 0.00 543Removed from experiment on day 14th after primary infection Mean 0.003.90 5.35 2.55 1.29 0.00 SD 0.00 0.95 1.23 2.39 1.84 0.00

It is to be understood that, while the methods and compositions ofmatter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the methods andcompositions of matter. Other aspects, advantages, and modifications arewithin the scope of the following claims.

Disclosed are methods and compositions that can be used for, can be usedin conjunction with, can be used in preparation for, or are products ofthe disclosed methods and compositions. These and other materials aredisclosed herein, and it is understood that combinations, subsets,interactions, groups, etc. of these methods and compositions aredisclosed. That is, while specific reference to each various individualand collective combinations and permutations of these compositions andmethods may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particularcomposition of matter or a particular method is disclosed and discussedand a number of compositions or methods are discussed, each and everycombination and permutation of the compositions and the methods arespecifically contemplated unless specifically indicated to the contrary.Likewise, any subset or combination of these is also specificallycontemplated and disclosed.

1-17. (canceled)
 18. A PRRSV-CON polypeptide having at least 95%sequence identity to a sequence selected from the group consisting ofSEQ ID NO:29, 31, 33, and
 35. 19. The polypeptide of claim 18, having atleast 99% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO:29, 31, 33, and
 35. 20. The polypeptide of claim18, having a sequence selected from the group consisting of SEQ IDNO:29, 31, 33, and
 35. 21. The polypeptide of claim 20, wherein thepolypeptide is encoded by a nucleic acid, respectively, having asequence selected from the group consisting of SEQ ID NOs:28, 30, 32,and
 34. 22. A virus particle comprising the PPRSV-CON polypeptide ofclaim
 18. 23. A composition comprising the polypeptide of claim 18 and apharmaceutically acceptable carrier.
 24. A composition comprising thevirus particle of claim 22 and a pharmaceutically acceptable carrier.25. The composition of claim 23, further comprising an adjuvant.
 26. Amethod for eliciting an immune response to PPRSV in a porcine,comprising administering, to a porcine: an effective amount of thepolypeptide of claim
 18. 27. The method of claim 26, wherein theadministration is selected from the group consisting of intramuscularly,intraperitoneally, and orally.
 28. A method for treating or preventingPPRS in a porcine, comprising administering, to a porcine: an effectiveamount of the polypeptide of claim
 18. 29. The method of claim 28,wherein the administration is selected from the group consisting ofintramuscularly, intraperitoneally, and orally.